Difference between revisions of "Bezuidenhout 2016a Abstract MitoFit Science Camp 2016"

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Analysis and comparison of mitochondrial oxygen dependence with two respiration media, Buffer ZCtlCr + Blebbistatin and MiR06Cr, in permeabilized mouse skeletal muscle fibers.

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Bezuidenhout N, Doerrier C, Droescher S, Ojuka E, Gnaiger E (2016)

Event: MitoFit Science Camp 2016 Kuehtai AT

It has been reported that the oxygen (O₂) dependence of mitochondrial respiration in permeabilized fibers (Pfi) is ~100-fold higher compared to isolated mitochondria [1]. Pfi are sensitive to low oxygen supply due to diffusion restrictions that limit the supply of oxygen to the core of the fiber bundle. Therefore, hyperoxic conditions (oxygen range 400-250 μM) have been employed to counteract this limitation. Further studies have shown that the addition of a myosin II-specific inhibitor, blebbistatin (BLEB) in the respiration medium reduces this sensitivity and allows the study of ADP kinetics in Pfi at normoxic oxygen levels (250-200 μM). Moreover, it has been described that the use of BLEB prevents fiber contraction and yields high Km values for ADP [2].

In the present study, high-resolution respirometry (HRR) was used to test ADP respiratory kinetics in skeletal muscle Pfi from wild-type C57BL/6 mice under three different oxygen regimens: hypoxia (100-80 μM), normoxia (250-200 μM) and hyperoxia (400 μM) comparing two respiration media: Buffer Z with catalase (Ctl), creatine (Cr) and BLEB (BufferZCtlCr+BLEB) and MiR06+Cr (MiR06Cr) at a physiological temperature of 37 °C. Data were normalized to wet weight (ww) (mg of tissue), complex IV (COX) and citrate synthase (CS) activities.

In the present study, we found no differences in O₂ flux when compared between Buffer ZCtlCr + BLEB and MiR06Cr. Our results show that different O₂ regimens as hypoxic and normoxic conditions into the O2k-chamber exhibit a relevant underestimation of OXPHOS capacity due to the oxygen limitation in both media (BufferZCtlCr+BLEB and MiR06Cr). It would therefore appear that the use of BufferZCtlCr with the addition of BLEB does not alleviate O₂ dependency in permeabilized skeletal muscle fibers.

• O2k-Network Lab: ZA Cape Town Ojuka EO, AT Innsbruck Gnaiger E, AT Innsbruck OROBOROS

Labels: MiParea: Respiration

Stress:Oxidative stress;RONS Organism: Mouse Tissue;cell: Skeletal muscle Preparation: Permeabilized tissue

Regulation: ADP, Oxygen kinetics

HRR: Oxygraph-2k

MitoFit Science Camp 2016

Affiliations

1-Univ Cape Town, Dept Human Biol, South Africa; 2-OROBOROS INSTRUMENTS, Innsbruck, Austria; 3-D.Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria. - [email protected]

Figures

Figure 1:

References and Support

Scandurra FM, Gnaiger E (2010) Cell respiration under hypoxia: facts and artefacts in mitochondrial oxygen kinetics. Adv Exp Med Biol 662:7-25. »Bioblast link
Perry CG, Kane DA, Lin CT, Kozy R, Cathey BL, Lark DS, Kane CL, Brophy PM, Gavin TP, Anderson EJ, Neufer PD (2011) Inhibiting myosin-ATPase reveals a dynamic range of mitochondrial respiratory control in skeletal muscle. Biochem J 437:215-22. »Bioblast link

The project was supported by K-Regio project MitoFit, funded in part by the Tyrolian Government and the European Regional Development Fund (ERDF).

@@ Line 1: / Line 1: @@
 {{Abstract
 |title=Analysis and comparison of mitochondrial oxygen dependence with two respiration media, Buffer ZCtlCr + Blebbistatin and MiR06Cr, in permeabilized mouse skeletal muscle fibers.
 |authors=Bezuidenhout N, Doerrier C, Droescher S, Ojuka E, Gnaiger E
 |year=2016
 |event=MitoFit Science Camp 2016 Kuehtai AT
+|abstract=It has been reported that the oxygen (O<sub>2</sub>) dependence of mitochondrial respiration in permeabilized fibers (Pfi) is ~100-fold higher compared to isolated mitochondria [1]. Pfi are sensitive to low oxygen supply due to diffusion restrictions that limit the supply of oxygen to the core of the fiber bundle. Therefore, hyperoxic conditions (oxygen range 400-250 μM) have been employed to counteract this limitation. Further studies have shown that the addition of a myosin II-specific inhibitor, blebbistatin (BLEB) in the respiration medium reduces this sensitivity and allows the study of ADP kinetics in Pfi at normoxic oxygen levels (250-200 μM). Moreover, it has been described that the use of BLEB prevents fiber contraction and yields high Km values for ADP [2].
+In the present study, high-resolution respirometry (HRR) was used to test ADP respiratory kinetics in skeletal muscle Pfi from wild-type C57BL/6 mice under three different oxygen regimens: hypoxia (100-80 μM), normoxia (250-200 μM) and hyperoxia (400 μM) comparing two respiration media: Buffer Z with catalase (Ctl), creatine (Cr) and BLEB (BufferZCtlCr+BLEB) and MiR06+Cr (MiR06Cr) at a physiological temperature of 37 °C. Data were normalized to wet weight (ww) (mg of tissue), complex IV (COX) and citrate synthase (CS) activities.
+In the present study, we found no differences in O<sub>2</sub> flux when compared between Buffer ZCtlCr + BLEB and MiR06Cr. Our results show that different O<sub>2</sub> regimens as hypoxic and normoxic conditions into the O2k-chamber exhibit a relevant underestimation of OXPHOS capacity due to the oxygen limitation in both media (BufferZCtlCr+BLEB and MiR06Cr). It would therefore appear that the use of BufferZCtlCr with the addition of BLEB does not alleviate O<sub>2</sub> dependency in permeabilized skeletal muscle fibers.
+|mipnetlab=ZA Cape Town Ojuka EO, AT Innsbruck Gnaiger E, AT Innsbruck OROBOROS
 }}
 {{Labeling
+|area=Respiration
+|organism=Mouse
+|tissues=Skeletal muscle
+|preparations=Permeabilized tissue
+|injuries=Oxidative stress;RONS
+|topics=ADP, Oxygen kinetics
+|instruments=Oxygraph-2k
 |additional=MitoFit Science Camp 2016
 }}
@@ Line 11: / Line 27: @@
 -Univ Cape Town, Dept Human Biol, South Africa; 2-OROBOROS INSTRUMENTS, Innsbruck, Austria; 3-D.Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria. - [email protected]
+== Figures ==
+[[Image:MSPfi MiR06-BufferZ.png|left|600px]] Figure 1:
 == References and Support ==